Cancer is a major disease burden and one hope of changing this is to develop new treatments through a better understanding of the disease. Recent discoveries concerning the activities of short RNAs in mammals may provide both new insights and new treatments of cancer. Cancer is a disease of gene dysregulation caused by mutations and epigenetic changes. Short RNAs are now known to regulate genes at the levels of mRNA degradation, primarily by siRNAs, mRNA translation, primarily by microRNAs, and mRNA transcription, primarily by repeat-associated short interfering RNAs (rasiRNAs). Recent results from bioinformatic studies indicate that approximately 20% of all mammalian mRNAs are probably regulated by mi RNAs. Furthermore, there is strong and rapidly growing evidence suggesting that changes in miRNA regulation is related to malignant transformation and in fact could be a critical event in oncogenic transformation. Little is known about the potential roles of short RNAs in silencing transcription at the level of chromatin in these cells. It is possible that some of the epigenetic changes and genomic instability common of cancers could be directed by RNAi-related pathways. Part of the revolution of RNA interference is the ability to express short hairpin RNAs from vectors to generate siRNAs which silence a specific gene. First, in collaboration with the Jacks and Lees projects, we will develop lentivirus vectors expressing shRNAs in a regulated fashion using tet-activated Pol II transcription. Second, we will use a very sensitive cloning technology for short RNAs to analyze their expression in T-cell populations as they undergo development. The activities of specific miRNAs will be related to known developmental transitions in this defined pathway. Furthermore, the nature of short RNAs in T-cell lymphomas and other tumor cells will be investigated by collaboration with the other projects. The silencing of repetitive sequences by both chromatin modification and DNA methylation is frequently observed in embryonic stem (ES) cells. We will clone short RNAs from ES cells which have been induced to express high levels of RNA from repetitive sequences to investigate the role of short RNAs in gene silencing at the level of transcription. The relationship between the resilencing of repetitive and unique sequences in these ES cells and the sequences of cloned short RNAs will be investigated. Furthermore, the dependence of these processes on Dicer, Argonaute and other RNAi-related genes will be determined. We will also determine the function of a cluster of miRNAs that is exclusively expressed in ES cells and embryonic tissue. Finally, we have found that both the retinoblastoma (Rb) pathway and the RNAi pathway regulate post-mitotic nuclear division in the intestinal epithelium of C. elegans. We will investigate whether related pathways are important for regulation of cell division in mammalian cells.